ABSTRACT
Recent in silico analysis has revealed the presence of a group of proteins in pro and lower eukaryotes, but not in Man, that show extensive amino acid sequence similarity to known O(6)-alkylguanine-DNA alkyltransferases, but where the cysteine at the putative active site is replaced by another residue, usually tryptophan. Here we review recent work on these proteins, which we designate as alkyltransferase-like (ATL) proteins, and consider their mechanism of action and role in protecting the host organisms against the biological effects of O(6)-alkylating agents, and their evolution. ATL proteins from Escherichia coli (eAtl, transcribed from the ybaz open reading frame) and Schizosaccharomyces pombe (Atl1) are able to bind to a range of O(6)-alkylguanine residues in DNA and to reversibly inhibit the action of the human alkyltransferase (MGMT) upon these substrates. Isolated proteins were not able to remove the methyl group in O(6)-methylguanine-containing DNA or oligonucleotides, neither did they display glycosylase or endonuclease activity. S. pombe does not contain a functional alkyltransferase and atl1 inactivation sensitises this organism to a variety of alkylating agents, suggesting that Atl1 acts by binding to O(6)-alkylguanine lesions and signalling them for processing by other DNA repair pathways. Currently we cannot exclude the possibility that ATL proteins arose through independent mutation of the alkyltransferase gene in different organisms. However, analyses of the proteins from E. coli and S. pombe, are consistent with a common function.
Subject(s)
Alkyl and Aryl Transferases/genetics , Alkyl and Aryl Transferases/metabolism , Alkyl and Aryl Transferases/chemistry , Alkylating Agents/toxicity , Amino Acid Sequence , Animals , DNA Modification Methylases/chemistry , DNA Modification Methylases/genetics , DNA Modification Methylases/metabolism , DNA Repair , DNA Repair Enzymes/chemistry , DNA Repair Enzymes/genetics , DNA Repair Enzymes/metabolism , Evolution, Molecular , Gene Deletion , Genes, Fungal , Humans , Molecular Sequence Data , O(6)-Methylguanine-DNA Methyltransferase/chemistry , O(6)-Methylguanine-DNA Methyltransferase/genetics , O(6)-Methylguanine-DNA Methyltransferase/metabolism , Phylogeny , Sequence Homology, Amino Acid , Species Specificity , Tumor Suppressor Proteins/chemistry , Tumor Suppressor Proteins/genetics , Tumor Suppressor Proteins/metabolismABSTRACT
Toxic and mutagenic O6-alkylguanine adducts in DNA are repaired by O6-alkylguanine-DNA alkyltransferases (MGMT) by transfer of the alkyl group to a cysteine residue in the active site. Comparisons in silico of prokaryotes and lower eukaryotes reveal the presence of a group of proteins [alkyltransferase-like (ATL) proteins] showing amino acid sequence similarity to MGMT, but where the cysteine at the putative active site is replaced by tryptophan. To examine whether ATL proteins play a role in the biological effects of alkylating agents, we inactivated the gene, referred to as atl1+, in Schizosaccharomyces pombe, an organism that does not possess a functional MGMT homologue. The mutants are substantially more susceptible to the toxic effects of the methylating agents, N-methyl-N-nitrosourea, N-methyl-N'nitro-N-nitrosoguanidine and methyl methanesulfonate and longer chain alkylating agents including N-ethyl-N-nitrosourea, ethyl methanesulfonate, N-propyl-N-nitrosourea and N-butyl-N-nitrosourea. Purified Atl1 protein does not transfer methyl groups from O6-methylguanine in [3H]-methylated DNA but reversibly inhibits methyl transfer by human MGMT. Atl1 binds to short single-stranded oligonucleotides containing O6-methyl, -benzyl, -4-bromothenyl or -hydroxyethyl-guanine but does not remove the alkyl group or base and does not cleave the oligonucleotide in the region of the lesion. This suggests that Atl1 acts by binding to O6-alkylguanine lesions and signalling them for processing by other DNA repair pathways. This is the first report describing an activity that protects S.pombe against the toxic effects of O6-alkylguanine adducts and the biological function of a family of proteins that is widely found in prokaryotes and lower eukaryotes.
Subject(s)
Alkyl and Aryl Transferases/metabolism , DNA Damage , DNA-Binding Proteins/metabolism , Schizosaccharomyces pombe Proteins/metabolism , Schizosaccharomyces/genetics , Alkyl and Aryl Transferases/genetics , Alkylating Agents/toxicity , DNA Repair , DNA-Binding Proteins/genetics , Gene Deletion , O(6)-Methylguanine-DNA Methyltransferase/metabolism , Oligonucleotides/chemistry , Schizosaccharomyces/drug effects , Schizosaccharomyces pombe Proteins/geneticsABSTRACT
The alkyltransferase-like (ATL) proteins contain primary sequence motifs resembling those found in DNA repair O(6)-alkylguanine-DNA alkyltransferase proteins. However, in the putative active site of ATL proteins, a tryptophan (W(83)) residue replaces the cysteine at the known active site of alkyltransferases. The Escherichia coli atl gene was expressed as a fusion protein and purified. Neither ATL nor C(83) or A(83) mutants transferred [(3)H] from [(3)H]-methylated DNA to themselves, and the levels of O(6)-methyl guanine (O(6)-meG) in substrate DNA were not affected by ATL. However, ATL inhibited the transfer of methyl groups to human alkyltransferase (MGMT). Inhibition was reduced by prolonged incubation in the presence of MGMT, again suggesting that O(6)-meG in the substrate is not changed by ATL. Gel-shift assays show that ATL binds to short single- or double-stranded oligonucleotides containing O(6)-meG, but not to oligonucleotides containing 8-oxoguanine, ethenoadenine, 5-hydroxycytosine or O(4)-methylthymine. There was no evidence of demethylation of O(6)-meG or of glycosylase or endonuclease activity. Overexpression of ATL in E.coli increased, or did not affect, the toxicity of N-methyl-N'-nitro-N-nitrosoguanidine in an alklyltransferase-proficient and -deficient strain, respectively. These results suggest that ATL may act as a damage sensor that flags O(6)-meG and possibly other O(6)-alkylation lesions for processing by other repair pathways.
